This project addresses the learning and motivational processes that influence ethanol-seeking behavior, including their underlying neurobiology. Special emphasis is placed on learning that results from the predictive relationship between environmental stimuli and ethanol's rewarding (or aversive) effects. Our central organizing hypothesis is that ethanol-predictive stimuli are critical for motivating, directing and maintainin ethanol-seeking behavior, including the phenomenon of relapse after a period of abstinence or after extinction. The strategy involves testing ethanol's motivational effects in mice using the conditioned place preference (CPP) procedure. Aim 1 will characterize brain systems underlying expression of ethanol-induced CPP using site-specific inactivation of 3 brain areas: the laterodorsal tegmental nucleus (LDTg), the lateral hypothalamus (LHA) and the arcuate hypothalamic nucleus (Arc). Using inhibitory or excitatory G-protein-coupled designer receptors exclusively activated by designer drugs (DREADDs), we then test the roles played during CPP expression by serial connection pathways between 5 brain areas (amygdala (Amy), bed nucleus of the stria terminalis (BNST), LDTg, LHA and Arc) and either the nucleus accumbens (Acb) or ventral tegmental area (VTA). Finally, to identify possible molecular mechanisms underlying ethanol CPP expression, we will also measure changes in relative phosphorylation of 4 proteins (GluA1, GluA2, PSD-95 and stargazin) linked to the glutamatergic AMPA receptor in VTA. Aim 2 uses conceptually similar approaches to determine whether the 5 brain areas tested in Aim 1 are also involved in ethanol CPP acquisition and to test the roles played by each of the proposed serial connection pathways (Amy-Acb; BNST-VTA; LDTg-VTA; LHA-VTA; Arc-VTA) in CPP acquisition. Aim 3 broadens our understanding of cue-elicited ethanol-seeking behavior by examining effects of acute ethanol withdrawal on ethanol-induced CPP. Cue-ethanol pairings that occur during acute withdrawal may be most relevant to understanding processes that facilitate the transition from episodic to chronic ethanol consumption in alcoholics. More specifically, Aim 3 will examine parametric determinants of the enhancement of ethanol CPP acquisition produced when conditioning trials occur during acute ethanol withdrawal (e.g., genotype, dose, post-withdrawal delay) as well as effects of acute withdrawal on CPP expression and extinction. Overall, these experiments should significantly improve our understanding of the behavioral processes and neurobiological systems that underlie the powerful effects of ethanol-predictive stimuli on behavior. By improving our understanding of these processes and systems, this project will help focus future research on specific brain and molecular mechanisms, aid in the development of beneficial treatments for alcohol-related disorders, and facilitate identification of more effective relapse prevention strategies. Our long- term goal is to improve our understanding of the behavioral and neurobiological processes that contribute to the etiology, maintenance, elimination and relapse of alcoholism.